The term “peptide immunotherapy” is used to describe the use of at least one peptide comprising a T cell epitope for the prevention or treatment of a disease, typically an autoimmune or an allergic disease. An example of an allergic disease is allergy to cats. Allergy to cats is typically characterised by allergic responses to one or more proteins present in cat dander, such as the protein Fel d 1.
A peptide used in peptide immunotherapy typically comprises a T cell epitope of a relevant autoantigen or allergen. Thus, for example, peptides comprising a T cell epitope of Fel d 1 are used to treat or prevent allergy to cats.
Where a peptide is to be used in peptide immunotherapy, there is a general need for it to be stable during storage and transport and to have a long shelf-life.
Background to Salt Forms of Peptides
In contrast to many low molecular drugs where the salt form can have a significant effect on their pharmaceutical, pharmacodynamic and pharmacokinetic behaviour, the various salts of peptides typically do not differ much with respect to these characteristics, are applied in the same manner and they exhibit essentially the same pharmacokinetic profile.
Most of the currently approved peptide pharmaceuticals, except for acidic or acid-labile peptides such as sincalide, are sold as acetate salts (acetates) (Vergote et al. 2009).
The first peptides used as drugs were prepared in solution and purified by counter-current distribution (CCD). CCD systems usually contain acetic acid and therefore it was logical to present the peptides purified using such systems as their acetate salts. Subsequently, when peptides were first synthesised by solid phase peptide synthesis (SPPS) in the 1980s, they were manufactured using Boc α-amino protecting group chemistry. This chemistry predicates the use of side chain protecting groups that require the use of anhydrous hydrogen fluoride for side chain deprotection and cleavage from the solid phase resin. Complete removal of residual fluoride ions from the peptides was necessary and not only was acetate a suitable molecule for replacement of the fluoride, but appropriate ion exchange resins were readily available.
With the introduction of Fmoc chemistry, side chain deprotection and cleavage from the resin could be achieved with the use of trifluoroacetic acid (TFA). The crude peptides resulting from the cleavage are typically purified by reverse phase liquid chromatography utilising elution systems that contain TFA as a modifier; following lyophilisation the purified peptides contain residual trifluoroacetate counterions. While some peptides, namely corticorelin (ovine) and Bivalirudin (Angiomax®) are available as trifluoroacetates (triflutate), ion exchange to switch the counterion to the acetate using appropriate resins is achieved readily and is usually undertaken since acetate is considered to be more acceptable from a toxicological perspective than trifluoroacetate (Hay, 2012).
The production and use of peptides as their acetate salts is advantageous for a number of reasons. Not only is acetate acceptable and compatible from a biological and toxicological perspective, but it is sufficiently volatile to allow removal of excess acetic acid during final lyophilisation of the peptide. The absolute peptide content is typically 10 to 20% higher when peptides are presented as acetates compared to when they are presented as trifluoroacetate salts due to the relative molecular weights of the two counterions. This has the potential to bring significant economic benefits although any savings gained from an increased peptide content may be offset to a degree by the costs associated with the additional ion exchange step required to convert the trifluoroacetate to the acetate form.
Due to the inherent differences in their primary sequences, there are no conditions that are universally optimum with respect to peptide stability. However, it is generally accepted that peptides typically exhibit maximal solution phase stability within the pH range 3 to 6 (Avanti, 2012), with deamidation being minimised within a pH range of 3 to 5. The use of acetate as a counterion facilitates the generation of solutions at this pH and the specific use of acetate matrices has been reported to improve the stability of peptides (Helm and Müller, 1990).
Consequently, commercially available peptides are typically produced as acetate salts unless there is a compelling reason to produce them as an alternative salt. This is confirmed, for example in Manufacturing Chemist (July/August 2012, p 40-41).
Alternative salt forms are required, or preferred, in certain circumstances, for instance, in the production of slow or controlled release preparations of peptides in biodegradable polymer formulations. WO2007/084460 (Quest Pharma) describes the preparation of salts of peptide agents using strong acids for incorporation into such formulations. The use of salts formed using strong acids relates to the neutralisation of basic functional groups contained within the peptides, i.e. at the N-terminus or within the side chains of arginine, lysine and histidine residues, through the formation of neutral salts using strong acids.
It is well recognised in the art that bioactive agents, i.e. peptides, containing basic amino functional groups interact with the biodegradable polymer and form conjugates with the polymer and/or its degradation products. These reactions can occur during preparation of the biodegradable polymer formulations, during storage thereafter and during degradation of the formulations in vivo. Neutralisation of the basic functional groups through formation of salts, such as hydrochlorides, using strong acids minimises or eliminates these reactions. Thus, the formation of salts with strong acids as described in this publication is specific to the use of peptides in biodegradable polymeric compositions.
A further example of the use of salts other than acetates is the use of HCl salts in minimising the conversion of N-terminal glutamic acid via a cyclization reaction to pyroglutamate/pyroglutamic acid (Beck et al., 2007). This particular use of non-acetate salts is specific to peptides having N-terminal glutamic acids.
Conversely, a number of disadvantages of working with strong acids are known. For instance, the use of hydrochloric acid to remove residual trifluoroacetate from peptides has been reported to result in degradation of the peptides (Andruschenko et al., 2007; Roux et al., 2008). The presence of trifluoroacetate interferes with the ability to characterize the physicochemical properties of peptides by infrared (IR) absorption spectroscopy; trifluoroacetate has a strong infrared (IR) absorption band at 1673 cm-1, significantly overlapping or even completely obscuring the amide I band of a peptide. The most convenient and widely used procedure involves lyophilizing the peptide several times in the presence of an excess of a stronger acid than trifluoroacetic acid (pKa approximately 0), i.e. generally hydrochloric acid (pKa=−7). However, this approach means working at pH<1 which can induce peptide degradation, most probably by acid hydrolysis; Andruschenko et al., (2007) reported peptide modification and reduction in thermal stability following the use of HCl to remove TFA. Interestingly, Roux et al. (2008) demonstrated the almost complete exchange of the trifluoroacetate counter-ion using acid weaker than trifluoroacetic acid, such as acetic acid (pKa=4.5) by means of an ion exchange resin as used routinely during conventional synthetic peptide manufacture, demonstrating the fact that strong acids are not required.
It is therefore currently the case that where pharmaceutically acceptable salt forms of peptides are required, it is routinely the acetate salts which are used. Stronger acids are associated with a number of potential disadvantages such as possible peptide degradation and thus are not routinely employed.